Project Summary Spermatogenesis is a classic stem cell dependent process essential for male reproduction. Undifferentiated spermatogonia with spermatogonial stem cells (SSCs or male germline stem cells, GSCs) at the foundation of the differentiation hierarchy undergo both self-renewal and differentiation to support lifelong production of sperm. Recent controversy exists regarding whether SSCs undergo unidirectional differentiation (?As model?) or interconvert between their progeny through dedifferentiation to form a heterogeneous pool of cells with stem cell function (?clone fragmentation model?). This debate is fundamental to our understanding of the hierarchy of male GSC differentiation. Moreover, it suggests an unrecognized yet critical physiological role of cellular dedifferentiation in normal stem cell maintenance. We have established a transgenic mouse line, in which the Stra8 promoter, whose activation is probably one of the earliest events during germline stem cell (GSC) differentiation of both sexes, drives GFP reporter expression (Stra8-GFP). In Stra8-GFP mice, we are able to separate spermatogonia into six populations at consecutive differentiation stages. These six populations include one for ?undifferentiated spermatogonia? (P1; potentially enriched for SSCs) defined by being negative for the Stra8-GFP reporter activity in addition to their cell surface phenotypes and five other populations for differentiating spermatogonia (P2 ? P4) and spermatocytes (P5 ? P6) defined by their distinctive Stra8-GFP reporter activity. In this study, we will use this novel approach to test our hypothesis that differentiating spermatogonia (P2 ? P4) undergo dedifferentiation back to undifferentiated stage (P1) to acquire stemness by inactivating Stra8 transcription under both regenerative and normal physiological conditions. We will: 1. Transplant each differentiating spermatogonia population (P2 ? P4) into chemotherapy-depleted testes to test their stemness and examine their dedifferentiation back to the undifferentiated stage (P1) under regenerative conditions. 2. Use genetic cell lineage tracing to permanently label differentiating spermatogonia and examine whether they generate SSCs that contributes to long-term spermatogenesis under normal physiological conditions. In addition to the basic science importance, our studies will aid the development of SSC transplantation therapy to improve and rescue fertility in clinical settings.